US10172848B2 - Gap junction-enhancing agents for treatment of necrotizing enterocolitis and inflammatory bowel disease - Google Patents

Gap junction-enhancing agents for treatment of necrotizing enterocolitis and inflammatory bowel disease Download PDF

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US10172848B2
US10172848B2 US13/921,865 US201313921865A US10172848B2 US 10172848 B2 US10172848 B2 US 10172848B2 US 201313921865 A US201313921865 A US 201313921865A US 10172848 B2 US10172848 B2 US 10172848B2
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David J. Hackam
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • This application relates to agents, particularly peptides and peptide analogs, that enhance the functionality of gap junctions between intestinal enterocytes, and which may be used to treat disorders associated with impaired interenterocyte gap junctions such as necrotizing enterocolitis and inflammatory bowel diseases. These gap junction enhancing peptides may also be used to reduce the risk of occurrence of these disorders.
  • Necrotizing enterocolitis is a leading cause of death and disability in premature infants. Patients that develop NEC do so suddenly and without warning, and upon surgical exploration of the abdomen, frequently demonstrate large regions of the intestine that are either dead or dying. In over half the cases, patients that develop NEC do not survive their disease, and in survivors, an additional third will develop long term complications related to the initial development of the disease.
  • NEC Necrotizing enterocolitis
  • NEC is characterized by impaired enterocyte migration along the crypt-villus axis.
  • the impaired enterocyte migration results in persistent mucosal defects, bacterial translocation, and the development of systemic sepsis which leads to death in many cases.
  • enterocytes migrate together as sheets, and that enterocyte migration is dependent upon intact inter-enterocyte communication via Cx43-mediated gap junctions (1, 2).
  • the release of the pro-inflammatory cytokine interferon gamma (IFN ⁇ ) plays a critical role in the impairment of mucosal healing in part by inhibiting gap junctions between enterocytes (1, 2).
  • IBD human inflammatory bowel disease
  • Gap junctions are intercellular channels that exist between adjacent cells which allow the transfer of small molecules between adjoining cells.
  • Each gap junction channel is comprised of a pair of hexameric arrays of individual subunits called connexins, of which the most widely expressed isoform is connexin-43 (Cx43; 2, 3, 4).
  • the function of gap junctions is regulated in part through phosphorylation of the individual connexin molecules, which serves to regulate the localization of the channels at the plasma membrane as well as to regulate the channel through gating (5, 6).
  • the present invention relates to methods of reducing the risk of occurrence of, and/or treating, necrotizing enterocolitis (“NEC”) or inflammatory bowel disease (“IBD”) comprising administering, to a subject in need of such treatment, an effective amount of a gap junction enhancing agent (“GJEA”), for example a peptide (“GJP”) or peptide analog (“GRA”).
  • GJEA gap junction enhancing agent
  • GJP peptide
  • GAA peptide analog
  • FIG. 1 Cx43 knockout “cko” mice were generated. RT-PCR demonstrated the absence of Cx43.
  • FIG. 2A-B Immunohistochemistry studies using fluorescently labeled antibodies directed toward Cx43 and actin demonstrate (A) the presence of both proteins in wild-type intestinal villi and (B) the absence of Cx43 in the villi of knock-out animals having the selective deletion of Cx43.
  • FIG. 3A-C (A) Photomicrograph of intestinal villi in a wild-type mouse. (B) Photomicrograph of intestinal villi in a Cx43 knockout mouse. (C) migration rate of enterocytes in wild-type versus Cx43 knockout (“ ⁇ IEC”).
  • FIG. 4 Absorption of fat, protein and glucose by WT versus Cx43 knockout mouse intestine.
  • FIG. 5A-H (A) Photomicrograph of intestinal tissue of a healthy wild-type mouse; (B) photomicrograph of intestinal tissue of a healthy wild-type mouse stained to show expression of inducible nitric oxide synthase (“iNOS”); (C) photomicrograph of intestinal tissue of a wild-type mouse having NEC; (D) photomicrograph of intestinal tissue of a wild-type mouse having NEC stained to show expression of iNOS; (E) photomicrograph of intestinal tissue of a Cx43 knockout mouse; (F) photomicrograph of intestinal tissue of a Cx43 knockout mouse stained to show expression of iNOS; (G) photomicrograph of intestinal tissue of a Cx43 knockout mouse having NEC; and (H) photomicrograph of intestinal tissue of a Cx43 knockout mouse with NEC stained to show expression of iNOS.
  • iNOS inducible nitric oxide synthase
  • FIG. 6A-B (A) Migration rate of enterocytes from either wild-type (“WT”) healthy (control) mice, wild-type mice having NEC, Cx43 knockout mice (“ ⁇ IEC”), or Cx43 mice having NEC. (B) Cytokine levels of either wild-type or Cx43 knockout mice (“Cx43 ⁇ IEC ”), having NEC.
  • FIG. 7A-B Cx43 expression in (A) premature (gestation day 15.5) or (B) postnatal (day 10) murine intestinal cells, as shown using a fluorescent anti-Cx43 antibody.
  • FIG. 8A-E (A) Photomicrograph of control murine intestine.
  • B Photomicrograph of murine intestine after exposure to adenoviral vector carrying Green Fluorescent Protein (“GFP”) gene.
  • C Photomicrograph of murine intestine after exposure to adenoviral vector carrying GFP and dominant negative Cx43 mutant protein (“dnCx43”).
  • D Expression of GFP in murine intestine in the presence (“DN”) or absence (“WT”) of dnCx43.
  • E Colitis severity in murine intestine in the presence (“DN”) or absence (“WT”) of dnCx43.
  • FIG. 9A-C Surface mucosa of murine intestine, either (A) control; (B) after exposure to adenoviral vector carrying GFP; or (C) after exposure to adenoviral vector carrying GFP and dnCx43.
  • FIG. 10A-B IEC-6 enterocytes were treated with the concentrations of phorbol myristate acetate (“PMA”) indicated for one hour and then evaluated for the extent of enterocyte gap junction communication by confocal based dye transfer. The untreated cells represent the 100% dye transfer. *p ⁇ 0.05 versus untreated cells.
  • IEC-6 cells were treated with the concentration of PMA indicated and assessed for their ability to migrate into a scraped wound over 20 hours. *p ⁇ 0.05 vs. untreated cells. Representative of two separate experiments.
  • PMA phorbol myristate acetate
  • Gap junction enhancing agents include gap junction enhancing peptides (“GJPs”) and peptide analogs (“GJPAs”) as well as other compounds such as pharmacologic agents.
  • Non-limiting examples of pharmacologic agents that are GJEAs include phorbol myristate acetate (“PMA”) and quinoline derivatives as described in United States Patent Publication No. 20090143425, such as, but not limited to, primaquine, mefloquine, PQ2, PQ3, PQ4, PQ5, PQ6, PQ7, PQ8 and/or the compound PQ1:
  • PMA phorbol myristate acetate
  • Gap Junction enhancing peptides that may be used according to the invention include but are not limited to peptides listed in United States Patent Application Publication No. 20090075291 by Delmar et al., which is incorporated by reference in its entirety herein, including: DVPGRDPGYIKGGGSAHARVPFYSHSLNRNRKPSLYQ (SEQ ID NO:1); EIQPRSPLMFSGGGSAHARVPFYSHSAKEARWPRAHR (SEQ ID NO:2); GIAAREPNSHDGGGSAHARVPFYSHSRDLWRKPAKSL (SEQ ID NO:3); WEEPRRPFTMSGGGSAETHARVPFYSHSPMRHRLPGVHL (SEQ ID NO:4); SDDLRSPQLHNGGGSAVPFYSHSHMVRRKPRNPR (SEQ ID NO:5); GHLHLRVPTLKM (SEQ ID NO:6); EFIRSPHSVDWL (SEQ ID NO:7); SQSRNPPMPPPR (SEQ ID NO:1)
  • GJPA is Compound ZP123 (Rotigaptide (2R, 4S)-1-[(2R)-1-[(2R)-2-acetamido-3-(4-hydroxyphenyl)propanoyl]pyrrolidine-2-carbonyl]-N-[2-[[(2R)-1-[(2-amino-2-oxoethyl)amino]-1-oxopropan-2-yl]amino]-2-oxoethyl]-4-hydroxypyrrolidine-2-carboxamide), having the following formula:
  • Rotigaptide which enhances gap junction connectivity through increased activation of Cx43.
  • the prototypical compound, Rotigaptide is stable, has a long half-life, and is in clinical trials as an anti-arrhythmic agent in adults, based upon its documented ability to enhance gap junction connectivity.
  • GJPs include pharmacophores of Cx43 based upon the “RXP” series of Cx43-binding peptides. These pharmacophores bind to the carboxyl terminal of Cx43, and share a 34-aa peptide (RXP-E (SEQ ID NO:27)) sequence. Two representatives of this family are a cyclized heptapeptide (called CyRP-71) and a linear octapeptide of sequence RRNYRRNY (SEQ ID NO:28).
  • GJP Gly-Ala-Gly-Hyp-Pro-Tyr-NH 2
  • AAP10 Gly-Ala-Gly-Hyp-Pro-Tyr-NH 2
  • AAP10 Gly-Ala-Gly-Hyp-Pro-Tyr-NH 2
  • related sequences such as but not limited to Gly-Pro-Hyp-Gly-Ala-Gly (SEQ ID NO:30) or cyclo[CF(3)C(OH)-Gly-Ala-Gly-Hyp-Pro-Tyr (SEQ ID NO:31).
  • the present invention further provides for peptides that are between 4 and 100, or between 4 and 50, or between 6 and 100, or between 6 and 50, or between 8 and 100, or between 8 and 50, or between 10 and 100, or between 10 and 50, or between 15 and 35, amino acids long and comprise one or more peptide having SEQ ID NO:1-31.
  • the present invention further provides for peptides that are between 4 and 100, or between 4 and 50, or between 6 and 100, or between 6 and 50, or between 8 and 100, or between 8 and 50, or between 10 and 100, or between 10 and 50, or between 15 and 35, amino acids long and comprise one or more peptide having a sequence which differs from any of SEQ ID NO:1-31 by no more than one amino acid and has a gap junction enhancing activity that is at least 80 percent of a peptide lacking the difference in sequence.
  • GJPA peptide analogs
  • GAP-134 i.e. (2S,4R)-1-(2-aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic acid
  • GAP-134 acts by enhancing gap junction conductance without effects on other ion channels, without any apparent changes in transcription or distribution of Cx43.
  • This compound has a shorter half-life than ZP123, which may decrease its effectiveness, but also may limit potential toxicity;
  • GJEA, GJP or GJPA ability of a GJEA, GJP or GJPA to enhance gap junction communication between enterocytes may be determined by any method known in the art, including in vitro and in vivo studies.
  • gap junction intercellular communication (GJIC) studies which assess the ability of a GJEA, GJP or GJPA to enhance gap junction communication between enterocytes, may be performed in vitro in cultured enterocytes, using live cell confocal microscopy and fluorescence recovery after photobleaching (FRAP), as in (1, 2).
  • FRAP live cell confocal microscopy and fluorescence recovery after photobleaching
  • cells may be plated to confluence, and treated with a GJEA, GJP or GJPA for times between 1-4 hours, and the degree of gap junction intercellular communication may be determined using confocal based fluorescence recovery after photobleaching (FRAP), which measures the movement of a fluorescent tracer through gap junctions into an area that has been previously photobleached using a laser, such that the rate and extent to which the photobleached cells fill with the fluorescent dye (termed the “fluorescence recovery”) may provide a direct measure of gap junction activity.
  • FRAP confocal based fluorescence recovery after photobleaching
  • a second non-limiting example of a method for assessing the ability of a GJEA, GJP or GJPA to enhance gap junction communication between enterocytes is single cell microinjection (2), which allows the detection of the extent to which a detectable tracer, for example the 0.4 kilodalton fluorescent gap junction tracer Lucifer yellow, passes from an injected cell to adjacent cells through gap junctions.
  • a detectable tracer for example the 0.4 kilodalton fluorescent gap junction tracer Lucifer yellow
  • IEC-6 cells may be used for such studies, as they represent a well validated culture model of the intestine.
  • Non-limiting examples of other cell lines that may be used include HT-29 and CaCO-2 cells.
  • the ability of a GJEA, GJP or GJPA to treat NEC may be assessed in vivo by the following study in mice.
  • the ability of a GJEA, GJP or GJPA to treat IBD may be assessed in vivo by the following study in mice.
  • the foregoing methods may be used to determine whether a particular concentration of a GJEA, GJP or GJPA is able to enhance gap junction communication between enterocytes, to reduce the incidence of NEC, to treat NEC, to reduce the incidence of IBD, or to treat IBD.
  • aGJEA, GJP or GJPA enhances (increases the rate or amount of) communication between adjacent enterocytes by at least about 10 percent, or at least about 20 percent, or at least about 30 percent, or at least about 40 percent, or at least about 50 percent, or at least about 60 percent, or at least about 70 percent, or at least about 80 percent, or at least about 100 percent, relative to the amount of communication under control conditions (e.g. the absence of the GJEA, GJP or GJPA).
  • the present invention provides for a pharmaceutical composition
  • a pharmaceutical composition comprising one or more GJEA, GJP or GJPA in a pharmaceutically suitable carrier.
  • Said pharmaceutical composition may be in solid or liquid form.
  • a pharmaceutical composition may comprise GJEA, GJP or GJPA which is optionally lyophilized or comprised in micelles or microspheres.
  • a pharmaceutical composition may comprise GJEA, GJP or GJPA in a solvent such as but not limited to water, saline, and/or a physiologic buffer, for example, but not limited to, a phosphate buffer, an acetate buffer, a carbonate buffer, a glutamate buffer, a glycinate buffer, a histidine buffer, a lactate buffer, a succinate buffer, a maleate buffer, a tartrate buffer, a Tris buffer, or a citrate buffer.
  • a solvent such as but not limited to water, saline, and/or a physiologic buffer, for example, but not limited to, a phosphate buffer, an acetate buffer, a carbonate buffer, a glutamate buffer, a glycinate buffer, a histidine buffer, a lactate buffer, a succinate buffer, a maleate buffer, a tartrate buffer, a Tris buffer, or a citrate buffer.
  • Non-limiting examples of other ingredients which may optionally be included in pharmaceutical compositions of the invention include albumin, ascorbic acid, sodium bisulphite, sodium metabisulphite, sodium sulphite, thioglycerolm thioglycolic acid, cysteine, ethylene diametetraacetic acid, citric acid/sodium citrate, ethylene glycol, glycerol, glucose, dextran, and/or a surfactant, for example sodium dodecyl sulfate, Polysorbate 80, and/or Polysorbate 20.
  • one or more GJEA, GJP or GJAP may be comprised in an infant nutritional formula, considered a pharmaceutical formulation and also a so-called “nutriceutical” formulation, which may be administered to an infant suffering from NEC or at risk of developing NEC to treat or reduced the risk of NEC in the infant.
  • infant nutritional formula may, for example and not by way of limitation, further comprise one or more of casein, whey protein, soy lecithin, lactose, dextrose, sodium chloride, potassium chloride, calcium carbonate, ferrous sulfate, ascorbic acid, vitamin A, vitamin B6, vitamin B12, vitamin D3, thiamine, vitamin E, and/or vitamin K.
  • the present invention provides for a method of treating NEC in a subject in need of such treatment comprising administering to the subject an effective amount of a GJEA, GJP or GJPA.
  • the present invention provides for a method of treating IBD in a subject in need of such treatment comprising administering to the subject an effective amount of a GJEA, GJP or GJPA.
  • the present invention provides for a method of reducing the risk of occurrence of NEC in a subject in need of such treatment comprising administering to the subject an effective amount of a GJEA, GJP or GJPA.
  • the present invention provides for a method of reducing the risk of occurrence of IBD in a subject in need of such treatment comprising administering to the subject an effective amount of a GJEA, GJP or GJPA.
  • a subject may be a human or non-human subject, including but not limited to a dog, cat, rodent, cow, sheep, goat, horse, or non-human primate.
  • the subject is a human infant.
  • the subject is a human infant born after less than 40 weeks or less than 37 weeks or less than 30 weeks or less than 25 weeks gestation.
  • IBD refers to disorders including but not limited to Crohn's disease, ulcerative colitis, lymphocytic colitis, ischemic colitis, Behçet's disease, diversion colitis, and irritable bowel syndrome.
  • a GJEA, GJP and/or GJPA for example as comprised in a pharmaceutical formulation, may be administered by any route known in the art, including but not limited to oral, intravenous, intraperitoneal, inhalation (nasal and pulmonary), subcutaneous, intramuscular, or rectal. It may be desirable to promote local rather than systemic delivery of a GJEA, GJP or GJPA so as to limit the effect of the agent on gap junctions outside the intestine, for example by selecting a method of administration such as oral or rectal and/or by providing a sustained release formulation.
  • Non-limiting examples of dosages include an amount that results in a local concentration at the intestinal mucosa between 10 and 200 nMol/L, or between 0.125 and 1 mM, or a dosage from 0.05 to 10 mg/kg or between 0.05 and 5 mg/kg or between 0.1 and 1 mg/kg. Dosages may, in non-limiting embodiments, be administered once, twice, three or four times daily, or every other day, or once a week, or once every two weeks, or once a month.
  • Treating means reducing the objective and/or subjective symptoms or signs of the disease being treated, including but not limited to one or more of: a decrease in intestinal tissue viability, malabsorption, diarrhea, bleeding, loss of electrolytes, return to normal activities of daily living and pain. In non-limiting embodiments the reduction is of a magnitude of at least about 20 percent or at least about 30 percent or at least about 40 percent or at least about 50 percent.
  • reducing the risk” of occurrence means a reduction in risk of at least about 10 percent or at least about 20 percent or at least about 30 percent or at least about 40 percent or at least about 50 percent.
  • Enterocyte-specific Cx43 knockout mice were generated using the Cre/loxP system, where Cx43 loxp/loxp mice were generated and crossed with villin-cre mice. The effectiveness of the “knockout” was documented by RT-PCR ( FIG. 1 ). As further confirmation, intestinal tissue of these mice was stained with detectable antibodies directed toward either Cx43 or actin, and the relative absence of Cx43 was apparent ( FIG. 2B ).
  • Enterocytes from the knockout mice were observed to be morphologically and functionally impaired relative to normal enterocytes. As shown in FIGS. 3B and 3A , respectively, the intestinal villi in the Cx43 knockout animals were substantially shortened compared with control intestine. Moreover, enterocytes from the knockout animals (“ ⁇ IEC”) demonstrated lower migration rates ( FIG. 3C ). As shown in FIG. 4 , Cx43 knockout mice were less able to absorb fat from their diet.
  • Enterocyte-specific Cx43 knockout mice were observed to manifest increased susceptibility to NEC.
  • NEC was induced in wild-type control mice and Cx43 knockout animals using a protocol that combines gavage feeding and hypoxia for 4 days.
  • the intestinal tissue of Cx43 knockout mice showed substantially more dramatic alteration relative to wild type (compare FIGS. 5G and 5C ), with substantially greater induction of inducible nitric oxide synthase (iNOS), a marker of inflammation (compare FIG. 5H with FIG. 5D ).
  • the enterocytes in the NEC-induced Cx43 knockout animals exhibited a slower migration rate ( FIG. 6A ) and inflammatory cytokine levels IL-6, TNF, and IL-1 were all substantially higher ( FIG. 6B ).
  • dnCx43 dominant negative mutant Cx43
  • Adenoviruses carrying dnCx43 with detectable marker GFP were constructed, as were adenoviruses carrying only GFP to serve as controls.
  • Virus was introduced into wild-type mice by rectal administration to produce GFP and GFP/dnCx43 animals.
  • Photomicrographs in FIGS. 8B and 8C show enterocytes of mice infected with adenovirus carrying GFP only or GFP and dnCx43, respectively, where the presence of fluorescence in these figures relative to FIG. 8A demonstrates successful viral infection.
  • IEC-6 enterocytes were treated with various concentrations of phorbol myristate acetate (“PMA”) for one hour and then evaluated for the extent of enterocyte gap junction communication by confocal based dye transfer. As shown in FIG. 10A , as concentrations of PMA increased, so did the extent of gap junction communication. Further, IEC-6 cells were treated with the concentration of PMA indicated and assessed for their ability to migrate into a scraped wound over 20 hours. As the concentration of PMA increased, the speed (rate) of enterocyte migration also increased, consistent with an association between gap junction enhancement and migration (and consequent healing) within the intestinal mucosa ( FIG. 10B ).
  • PMA phorbol myristate acetate

Abstract

The present invention relates to methods of reducing the risk of occurrence of, and/or treating, necrotizing enterocolitis (“NEC”) or inflammatory bowel disease (“IBD”) comprising administering, to a subject in need of such treatment, an effective amount of a gap junction enhancing agent (“GJEA”), for example a peptide (“GJP”) or peptide analog (“GJPA”). It is based, at least in part, on the discovery that greater functionality of gap junctions between enterocytes increases their rate of migration and reduces the severity of intestinal inflammation.

Description

PRIORITY
This application is a continuation of International Application No. PCT/US2011/066861, filed Dec. 22, 2011, and claims priority to U.S. Provisional Application No. 61/426,162, filed Dec. 22, 2010, the contents of which are expressly incorporated by reference herein.
SEQUENCE LISTING
The specification further incorporates by reference the Sequence Listing submitted herewith via EFS on Sep. 5, 2013. Pursuant to 37 C.F.R. § 1.52(e)(5), the Sequence Listing text file, identified as 0723960524_SL.txt, is 9,249 bytes and was created on Jul. 29, 2013. The Sequence Listing, electronically filed herewith, does not extend beyond the scope of the specification and thus does not contain new matter.
1. INTRODUCTION
This application relates to agents, particularly peptides and peptide analogs, that enhance the functionality of gap junctions between intestinal enterocytes, and which may be used to treat disorders associated with impaired interenterocyte gap junctions such as necrotizing enterocolitis and inflammatory bowel diseases. These gap junction enhancing peptides may also be used to reduce the risk of occurrence of these disorders.
2. BACKGROUND OF THE INVENTION
Necrotizing enterocolitis (NEC) is a leading cause of death and disability in premature infants. Patients that develop NEC do so suddenly and without warning, and upon surgical exploration of the abdomen, frequently demonstrate large regions of the intestine that are either dead or dying. In over half the cases, patients that develop NEC do not survive their disease, and in survivors, an additional third will develop long term complications related to the initial development of the disease. Currently, there is no specific therapy for NEC, and treatment involves the administration of broad spectrum antibiotics and surgical removal of the dead or dying intestine. Clearly, novel therapeutic approaches to this devastating disease are urgently needed.
In seeking to understand the pathogenesis of NEC, as well as to define novel therapeutic approaches for this disease, it was found that NEC is characterized by impaired enterocyte migration along the crypt-villus axis. The impaired enterocyte migration results in persistent mucosal defects, bacterial translocation, and the development of systemic sepsis which leads to death in many cases. In seeking to define the mechanisms that regulate enterocyte migration in both mice and humans, it was found that enterocytes migrate together as sheets, and that enterocyte migration is dependent upon intact inter-enterocyte communication via Cx43-mediated gap junctions (1, 2). Furthermore, the release of the pro-inflammatory cytokine interferon gamma (IFNγ) plays a critical role in the impairment of mucosal healing in part by inhibiting gap junctions between enterocytes (1, 2).
In other studies, it has been shown that human inflammatory bowel disease (“IBD”) is associated with impaired gap junctions within the intestinal mucosa (1, 2). Enterocyte migration is impaired in IBD just as it is in NEC.
Gap junctions are intercellular channels that exist between adjacent cells which allow the transfer of small molecules between adjoining cells. Each gap junction channel is comprised of a pair of hexameric arrays of individual subunits called connexins, of which the most widely expressed isoform is connexin-43 (Cx43; 2, 3, 4). The function of gap junctions is regulated in part through phosphorylation of the individual connexin molecules, which serves to regulate the localization of the channels at the plasma membrane as well as to regulate the channel through gating (5, 6).
3. SUMMARY OF THE INVENTION
The present invention relates to methods of reducing the risk of occurrence of, and/or treating, necrotizing enterocolitis (“NEC”) or inflammatory bowel disease (“IBD”) comprising administering, to a subject in need of such treatment, an effective amount of a gap junction enhancing agent (“GJEA”), for example a peptide (“GJP”) or peptide analog (“GRA”). It is based, at least in part, on the discovery that greater functionality of gap junctions between enterocytes increases their rate of migration and reduces the severity of intestinal inflammation.
4. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Cx43 knockout “cko” mice were generated. RT-PCR demonstrated the absence of Cx43.
FIG. 2A-B. Immunohistochemistry studies using fluorescently labeled antibodies directed toward Cx43 and actin demonstrate (A) the presence of both proteins in wild-type intestinal villi and (B) the absence of Cx43 in the villi of knock-out animals having the selective deletion of Cx43.
FIG. 3A-C. (A) Photomicrograph of intestinal villi in a wild-type mouse. (B) Photomicrograph of intestinal villi in a Cx43 knockout mouse. (C) migration rate of enterocytes in wild-type versus Cx43 knockout (“ΔIEC”).
FIG. 4. Absorption of fat, protein and glucose by WT versus Cx43 knockout mouse intestine.
FIG. 5A-H. (A) Photomicrograph of intestinal tissue of a healthy wild-type mouse; (B) photomicrograph of intestinal tissue of a healthy wild-type mouse stained to show expression of inducible nitric oxide synthase (“iNOS”); (C) photomicrograph of intestinal tissue of a wild-type mouse having NEC; (D) photomicrograph of intestinal tissue of a wild-type mouse having NEC stained to show expression of iNOS; (E) photomicrograph of intestinal tissue of a Cx43 knockout mouse; (F) photomicrograph of intestinal tissue of a Cx43 knockout mouse stained to show expression of iNOS; (G) photomicrograph of intestinal tissue of a Cx43 knockout mouse having NEC; and (H) photomicrograph of intestinal tissue of a Cx43 knockout mouse with NEC stained to show expression of iNOS.
FIG. 6A-B. (A) Migration rate of enterocytes from either wild-type (“WT”) healthy (control) mice, wild-type mice having NEC, Cx43 knockout mice (“ΔIEC”), or Cx43 mice having NEC. (B) Cytokine levels of either wild-type or Cx43 knockout mice (“Cx43ΔIEC”), having NEC.
FIG. 7A-B. Cx43 expression in (A) premature (gestation day 15.5) or (B) postnatal (day 10) murine intestinal cells, as shown using a fluorescent anti-Cx43 antibody.
FIG. 8A-E. (A) Photomicrograph of control murine intestine. (B) Photomicrograph of murine intestine after exposure to adenoviral vector carrying Green Fluorescent Protein (“GFP”) gene. (C) Photomicrograph of murine intestine after exposure to adenoviral vector carrying GFP and dominant negative Cx43 mutant protein (“dnCx43”). (D) Expression of GFP in murine intestine in the presence (“DN”) or absence (“WT”) of dnCx43. (E) Colitis severity in murine intestine in the presence (“DN”) or absence (“WT”) of dnCx43.
FIG. 9A-C. Surface mucosa of murine intestine, either (A) control; (B) after exposure to adenoviral vector carrying GFP; or (C) after exposure to adenoviral vector carrying GFP and dnCx43.
FIG. 10A-B. (A) IEC-6 enterocytes were treated with the concentrations of phorbol myristate acetate (“PMA”) indicated for one hour and then evaluated for the extent of enterocyte gap junction communication by confocal based dye transfer. The untreated cells represent the 100% dye transfer. *p<0.05 versus untreated cells. (B) IEC-6 cells were treated with the concentration of PMA indicated and assessed for their ability to migrate into a scraped wound over 20 hours. *p<0.05 vs. untreated cells. Representative of two separate experiments.
 5. DETAILED DESCRIPTION OF THE INVENTION
For clarity of description, and not by way of limitation, the detailed description of the invention is divided into the following subsections:
(i) gap junction enhancing agents;
(ii) assay methods;
(iii) pharmaceutical compositions; and
(iv) methods of treatment.
5.1 Gap Junction Enhancing Agents
Gap junction enhancing agents (“GJEAs”) include gap junction enhancing peptides (“GJPs”) and peptide analogs (“GJPAs”) as well as other compounds such as pharmacologic agents.
Non-limiting examples of pharmacologic agents that are GJEAs include phorbol myristate acetate (“PMA”) and quinoline derivatives as described in United States Patent Publication No. 20090143425, such as, but not limited to, primaquine, mefloquine, PQ2, PQ3, PQ4, PQ5, PQ6, PQ7, PQ8 and/or the compound PQ1:
Figure US10172848-20190108-C00001
Gap Junction enhancing peptides (“GJPs”) that may be used according to the invention include but are not limited to peptides listed in United States Patent Application Publication No. 20090075291 by Delmar et al., which is incorporated by reference in its entirety herein, including: DVPGRDPGYIKGGGSAHARVPFYSHSLNRNRKPSLYQ (SEQ ID NO:1); EIQPRSPLMFSGGGSAHARVPFYSHSAKEARWPRAHR (SEQ ID NO:2); GIAAREPNSHDGGGSAHARVPFYSHSRDLWRKPAKSL (SEQ ID NO:3); WEEPRRPFTMSGGGSAETHARVPFYSHSPMRHRLPGVHL (SEQ ID NO:4); SDDLRSPQLHNGGGSAVPFYSHSHMVRRKPRNPR (SEQ ID NO:5); GHLHLRVPTLKM (SEQ ID NO:6); EFIRSPHSVDWL (SEQ ID NO:7); SQSRNPPMPPPR (SEQ ID NO:8); RRPPYRVPPKLF (SEQ ID NO:9); SLYERHPASTYP (SEQ ID NO:10); HTVSRRPLPSSG (SEQ ID NO:11); RHTHGNLLRFPP (SEQ ID NO:12); RNNLNQTYPERR (SEQ ID NO:13); YSLLPVRPVALT (SEQ ID NO:14); RKPTQSLPTRLV (SEQ ID NO:15); TRRPHKMRSDPL (SEQ ID NO:16); TLTWHTKTPVRP (SEQ ID NO:17); SRQFLHSLDRLP (SEQ ID NO:18); HLHHHLDHRPHR (SEQ ID NO:19); QTPYQARLPAVA (SEQ ID NO:20); WHPHRHHHLQWD (SEQ ID NO:21); RRKPRRKP (SEQ ID NO:22); RNPRNP (SEQ ID NO:23); RRKP (SEQ ID NO:24); RRNP (SEQ ID NO:25); RNP or SDDLRSPQLHNHMVRRKPRNPR (SEQ ID NO:26), and also include other peptides and peptide derivatives that enhance gap junction communication between enterocytes.
One non-limiting example of a GJPA is Compound ZP123 (Rotigaptide (2R, 4S)-1-[(2R)-1-[(2R)-2-acetamido-3-(4-hydroxyphenyl)propanoyl]pyrrolidine-2-carbonyl]-N-[2-[[(2R)-1-[(2-amino-2-oxoethyl)amino]-1-oxopropan-2-yl]amino]-2-oxoethyl]-4-hydroxypyrrolidine-2-carboxamide), having the following formula:
Figure US10172848-20190108-C00002
which enhances gap junction connectivity through increased activation of Cx43. The prototypical compound, Rotigaptide, is stable, has a long half-life, and is in clinical trials as an anti-arrhythmic agent in adults, based upon its documented ability to enhance gap junction connectivity.
Further non-limiting examples of GJPs include pharmacophores of Cx43 based upon the “RXP” series of Cx43-binding peptides. These pharmacophores bind to the carboxyl terminal of Cx43, and share a 34-aa peptide (RXP-E (SEQ ID NO:27)) sequence. Two representatives of this family are a cyclized heptapeptide (called CyRP-71) and a linear octapeptide of sequence RRNYRRNY (SEQ ID NO:28).
Another non-limiting example of a GJP that may be used according to the present invention is Gly-Ala-Gly-Hyp-Pro-Tyr-NH2 (“AAP10”; SEQ ID NO:29) and related sequences such as but not limited to Gly-Pro-Hyp-Gly-Ala-Gly (SEQ ID NO:30) or cyclo[CF(3)C(OH)-Gly-Ala-Gly-Hyp-Pro-Tyr (SEQ ID NO:31).
The present invention further provides for peptides that are between 4 and 100, or between 4 and 50, or between 6 and 100, or between 6 and 50, or between 8 and 100, or between 8 and 50, or between 10 and 100, or between 10 and 50, or between 15 and 35, amino acids long and comprise one or more peptide having SEQ ID NO:1-31.
The present invention further provides for peptides that are between 4 and 100, or between 4 and 50, or between 6 and 100, or between 6 and 50, or between 8 and 100, or between 8 and 50, or between 10 and 100, or between 10 and 50, or between 15 and 35, amino acids long and comprise one or more peptide having a sequence which differs from any of SEQ ID NO:1-31 by no more than one amino acid and has a gap junction enhancing activity that is at least 80 percent of a peptide lacking the difference in sequence.
The present invention further provides for peptide analogs (“GJPA”) such as compound GAP-134 (i.e. (2S,4R)-1-(2-aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic acid) or other peptide analogs that enhance gap junction communication between enterocytes. Without being bound by any theory, GAP-134 acts by enhancing gap junction conductance without effects on other ion channels, without any apparent changes in transcription or distribution of Cx43. This compound has a shorter half-life than ZP123, which may decrease its effectiveness, but also may limit potential toxicity;
5.2 Assay Methods
The ability of a GJEA, GJP or GJPA to enhance gap junction communication between enterocytes may be determined by any method known in the art, including in vitro and in vivo studies.
As a non-limiting example, gap junction intercellular communication (GJIC) studies, which assess the ability of a GJEA, GJP or GJPA to enhance gap junction communication between enterocytes, may be performed in vitro in cultured enterocytes, using live cell confocal microscopy and fluorescence recovery after photobleaching (FRAP), as in (1, 2). For example, cells may be plated to confluence, and treated with a GJEA, GJP or GJPA for times between 1-4 hours, and the degree of gap junction intercellular communication may be determined using confocal based fluorescence recovery after photobleaching (FRAP), which measures the movement of a fluorescent tracer through gap junctions into an area that has been previously photobleached using a laser, such that the rate and extent to which the photobleached cells fill with the fluorescent dye (termed the “fluorescence recovery”) may provide a direct measure of gap junction activity.
A second non-limiting example of a method for assessing the ability of a GJEA, GJP or GJPA to enhance gap junction communication between enterocytes is single cell microinjection (2), which allows the detection of the extent to which a detectable tracer, for example the 0.4 kilodalton fluorescent gap junction tracer Lucifer yellow, passes from an injected cell to adjacent cells through gap junctions.
As one specific non-limiting example, IEC-6 cells may be used for such studies, as they represent a well validated culture model of the intestine. Non-limiting examples of other cell lines that may be used include HT-29 and CaCO-2 cells.
In certain non-limiting embodiments of the invention, the ability of a GJEA, GJP or GJPA to treat NEC may be assessed in vivo by the following study in mice.
    • a. Power analysis: Sample size estimates may be conducted using the nQuery Advisor 3.0 software (Statistical Solutions, Saugus, Mass.), with α=0.05, and differences in proportions between Groups averaging 0.22 with a variance of 0.029 and an effect size of 0.167 yielding 15 animals per group to attain 80% power. An estimated 20% of the animals would not be expected to survive, therefore the sample size should be increased to 18 to account for mortality loss. Experiments performed in triplicate with 18 mice per group, 4 groups per experiment=216 mice.
    • b. Induction of experimental NEC: NEC may be induced in 10-day old wild-type mice. Mice may be administered 15 g Similac 60/40 (Ross Pediatrics) in 75 mL of Esbilac canine milk replacer (Pet-Ag Inc) as well as hypoxia (5% oxygen for 2 minute prior to each feeding) twice daily for four days. Animals may be fed 200 microliters per 5 grams of mouse body weight by gavage over 2-3 minutes, using a 24-French angio-catheter which is placed into the mouse esophagus under direct vision. Samples of the terminal ileum 2 cm away from the ileocecal valve may then be harvested at day four for analysis. Control (i.e. non NEC) animals may remain with their mothers and receive breast milk.
    • c. Timing of administration of gap junction peptides: animals may be treated with GJEA, GJP or GJPA prior to the induction of experimental NEC; for example at varying doses, twice daily for either 1, 2, 3 or 4 days prior to the induction of NEC. Morbidity in animals that receive GJEA, GJP or GJPA alone should be assessed. Peptides may be administered via the i.p (intraperitoneal) or the p.o. (oral) route
    • d. Assessment of the severity of experimental NEC: The extent of NEC that develops may be assessed by measuring 1) histopathological evidence of mucosal damage. 2) serum IL-6 as determined by ELISA. 3) RT-PCR to assess the expression of IL-6, IL-8 and TNF-α in the mucosal scrapings, for example using mouse specific primers.
In certain non-limiting embodiments of the invention, the ability of a GJEA, GJP or GJPA to treat IBD may be assessed in vivo by the following study in mice.
    • a. Power analysis: Sample size estimates may be conducted using the nQuery Advisor 3.0 software (Statistical Solutions, Saugus, Mass.), with α=0.05, and differences in proportions between Groups averaging 0.22 with a variance of 0.029 and an effect size of 0.167 yielding 15 animals per group to attain 80% power. There is essentially no mortality in the colitis model, and the sample size may be 25 per group. Total mice: experiments in triplicate, 4 groups, 25 per group—300 mice.
    • b. Induction of experimental colitis: Colitis may be induced in 4 week old mice using the dextran sodium sulfate model. 4% DSS may be administered in the drinking water for 5 days, at which point 100% of mice develop inflammation of the mucosa of the colon, associated with infiltration of inflammatory cells.
    • c. Timing of administration of gap junction peptides: animals may be treated with GJEA, GJP or GJPA prior to the induction of colitis; animals may be administered GJEA, GJP or GJPA for varying doses, twice daily for either 1, 2, 3 or 4 days prior to the induction of colitis. Morbidity in animals that receive GJEA, GJP or GJPA alone should be assessed. GJEA, GJP or GJPA may be administered via the i.p or the p.o. route
    • d. Assessment of the severity of experimental colitis: The extent of colitis that develops may be assessed by measuring 1) histopathological evidence of mucosal damage. 2) serum IL-6 as determined by ELISA. 3) RT-PCR to assess the expression of IL-6, IL-8 and TNF-α in the mucosal scrapings, for example using mouse specific primers.
The foregoing methods may be used to determine whether a particular concentration of a GJEA, GJP or GJPA is able to enhance gap junction communication between enterocytes, to reduce the incidence of NEC, to treat NEC, to reduce the incidence of IBD, or to treat IBD.
In non-limiting embodiments of the invention, aGJEA, GJP or GJPA enhances (increases the rate or amount of) communication between adjacent enterocytes by at least about 10 percent, or at least about 20 percent, or at least about 30 percent, or at least about 40 percent, or at least about 50 percent, or at least about 60 percent, or at least about 70 percent, or at least about 80 percent, or at least about 100 percent, relative to the amount of communication under control conditions (e.g. the absence of the GJEA, GJP or GJPA).
5.3 Pharmaceutical Compositions
The present invention provides for a pharmaceutical composition comprising one or more GJEA, GJP or GJPA in a pharmaceutically suitable carrier.
Said pharmaceutical composition may be in solid or liquid form.
In non-limiting embodiments of the invention, a pharmaceutical composition may comprise GJEA, GJP or GJPA which is optionally lyophilized or comprised in micelles or microspheres.
In non-limiting embodiments of the invention, a pharmaceutical composition may comprise GJEA, GJP or GJPA in a solvent such as but not limited to water, saline, and/or a physiologic buffer, for example, but not limited to, a phosphate buffer, an acetate buffer, a carbonate buffer, a glutamate buffer, a glycinate buffer, a histidine buffer, a lactate buffer, a succinate buffer, a maleate buffer, a tartrate buffer, a Tris buffer, or a citrate buffer.
Non-limiting examples of other ingredients which may optionally be included in pharmaceutical compositions of the invention include albumin, ascorbic acid, sodium bisulphite, sodium metabisulphite, sodium sulphite, thioglycerolm thioglycolic acid, cysteine, ethylene diametetraacetic acid, citric acid/sodium citrate, ethylene glycol, glycerol, glucose, dextran, and/or a surfactant, for example sodium dodecyl sulfate, Polysorbate 80, and/or Polysorbate 20.
In a set of specific non-limiting embodiments of the invention, one or more GJEA, GJP or GJAP may be comprised in an infant nutritional formula, considered a pharmaceutical formulation and also a so-called “nutriceutical” formulation, which may be administered to an infant suffering from NEC or at risk of developing NEC to treat or reduced the risk of NEC in the infant. Such infant nutritional formula may, for example and not by way of limitation, further comprise one or more of casein, whey protein, soy lecithin, lactose, dextrose, sodium chloride, potassium chloride, calcium carbonate, ferrous sulfate, ascorbic acid, vitamin A, vitamin B6, vitamin B12, vitamin D3, thiamine, vitamin E, and/or vitamin K.
5.4 Methods of Treatment
In non-limiting embodiments, the present invention provides for a method of treating NEC in a subject in need of such treatment comprising administering to the subject an effective amount of a GJEA, GJP or GJPA.
In other non-limiting embodiments, the present invention provides for a method of treating IBD in a subject in need of such treatment comprising administering to the subject an effective amount of a GJEA, GJP or GJPA.
In other non-limiting embodiments, the present invention provides for a method of reducing the risk of occurrence of NEC in a subject in need of such treatment comprising administering to the subject an effective amount of a GJEA, GJP or GJPA.
In other non-limiting embodiments, the present invention provides for a method of reducing the risk of occurrence of IBD in a subject in need of such treatment comprising administering to the subject an effective amount of a GJEA, GJP or GJPA.
A subject may be a human or non-human subject, including but not limited to a dog, cat, rodent, cow, sheep, goat, horse, or non-human primate. In non-limiting embodiments the subject is a human infant. In non-limiting embodiments the subject is a human infant born after less than 40 weeks or less than 37 weeks or less than 30 weeks or less than 25 weeks gestation.
IBD as that term is used herein refers to disorders including but not limited to Crohn's disease, ulcerative colitis, lymphocytic colitis, ischemic colitis, Behçet's disease, diversion colitis, and irritable bowel syndrome.
A GJEA, GJP and/or GJPA, for example as comprised in a pharmaceutical formulation, may be administered by any route known in the art, including but not limited to oral, intravenous, intraperitoneal, inhalation (nasal and pulmonary), subcutaneous, intramuscular, or rectal. It may be desirable to promote local rather than systemic delivery of a GJEA, GJP or GJPA so as to limit the effect of the agent on gap junctions outside the intestine, for example by selecting a method of administration such as oral or rectal and/or by providing a sustained release formulation.
Non-limiting examples of dosages include an amount that results in a local concentration at the intestinal mucosa between 10 and 200 nMol/L, or between 0.125 and 1 mM, or a dosage from 0.05 to 10 mg/kg or between 0.05 and 5 mg/kg or between 0.1 and 1 mg/kg. Dosages may, in non-limiting embodiments, be administered once, twice, three or four times daily, or every other day, or once a week, or once every two weeks, or once a month.
“Treating” means reducing the objective and/or subjective symptoms or signs of the disease being treated, including but not limited to one or more of: a decrease in intestinal tissue viability, malabsorption, diarrhea, bleeding, loss of electrolytes, return to normal activities of daily living and pain. In non-limiting embodiments the reduction is of a magnitude of at least about 20 percent or at least about 30 percent or at least about 40 percent or at least about 50 percent.
In non-limiting embodiments, “reducing the risk” of occurrence means a reduction in risk of at least about 10 percent or at least about 20 percent or at least about 30 percent or at least about 40 percent or at least about 50 percent.
6. EXAMPLE 1: SUSCEPTIBILITY TO NEC OF CX43 KNOCKOUT MICE
Enterocyte-specific Cx43 knockout mice were generated using the Cre/loxP system, where Cx43 loxp/loxp mice were generated and crossed with villin-cre mice. The effectiveness of the “knockout” was documented by RT-PCR (FIG. 1). As further confirmation, intestinal tissue of these mice was stained with detectable antibodies directed toward either Cx43 or actin, and the relative absence of Cx43 was apparent (FIG. 2B).
Enterocytes from the knockout mice were observed to be morphologically and functionally impaired relative to normal enterocytes. As shown in FIGS. 3B and 3A, respectively, the intestinal villi in the Cx43 knockout animals were substantially shortened compared with control intestine. Moreover, enterocytes from the knockout animals (“ΔIEC”) demonstrated lower migration rates (FIG. 3C). As shown in FIG. 4, Cx43 knockout mice were less able to absorb fat from their diet.
Enterocyte-specific Cx43 knockout mice were observed to manifest increased susceptibility to NEC. NEC was induced in wild-type control mice and Cx43 knockout animals using a protocol that combines gavage feeding and hypoxia for 4 days. When exposed to the same conditions, the intestinal tissue of Cx43 knockout mice showed substantially more dramatic alteration relative to wild type (compare FIGS. 5G and 5C), with substantially greater induction of inducible nitric oxide synthase (iNOS), a marker of inflammation (compare FIG. 5H with FIG. 5D). The enterocytes in the NEC-induced Cx43 knockout animals exhibited a slower migration rate (FIG. 6A) and inflammatory cytokine levels IL-6, TNF, and IL-1 were all substantially higher (FIG. 6B).
The above findings support an association between NEC and Cx43 deficiency and gap junction dysfunction.
7. EXAMPLE 2: CX43 IN PREMATURE BOWEL
It was observed that Cx43 expression is reduced in the eneterocytes of mouse embryos on day e15.5 (FIG. 7A) relative to eneterocytes of mice at postnatal day 10 (FIG. 7B). As the incidence of NEC is increased in premature, versus full term, human infants, the observation of lower levels of Cx43 in the “premature” intestine is consistent with a model in which Cx43 deficiency and consequent gap junction dysfunction increases susceptibility to NEC.
8. EXAMPLE 3: HIGHER LEVELS OF CX43 CORRELATE WITH LESS COLITIS
Experiments were performed using adenovirus to introduce dominant negative mutant Cx43 (“dnCx43”) into murine enterocytes and thereby create a functional Cx43 deficiency. Adenoviruses carrying dnCx43 with detectable marker GFP were constructed, as were adenoviruses carrying only GFP to serve as controls. Virus was introduced into wild-type mice by rectal administration to produce GFP and GFP/dnCx43 animals. Photomicrographs in FIGS. 8B and 8C show enterocytes of mice infected with adenovirus carrying GFP only or GFP and dnCx43, respectively, where the presence of fluorescence in these figures relative to FIG. 8A demonstrates successful viral infection.
Next, colitis was induced in GFP, GFP/dnCx43 and control animals by administration of dextran sulphate sodium. Cx43 deficiency and consequent gap junction dysfunction and susceptibility to NEC. Visualization of the intestinal mucosa in these animals is shown in FIG. 9A-C. The most severe colitis was observed GFP/dn-Cx43 mice (FIGS. 8E and 9C).
According to these studies, the relatively greater amount of Cx43 in animals that had not received dnCx43 was protective against colitis.
9. EXAMPLE 4: GAP JUNCTION ENHANCER PMA PROMOTES ENTEROCYTE MIGRATION
IEC-6 enterocytes were treated with various concentrations of phorbol myristate acetate (“PMA”) for one hour and then evaluated for the extent of enterocyte gap junction communication by confocal based dye transfer. As shown in FIG. 10A, as concentrations of PMA increased, so did the extent of gap junction communication. Further, IEC-6 cells were treated with the concentration of PMA indicated and assessed for their ability to migrate into a scraped wound over 20 hours. As the concentration of PMA increased, the speed (rate) of enterocyte migration also increased, consistent with an association between gap junction enhancement and migration (and consequent healing) within the intestinal mucosa (FIG. 10B).
7. REFERENCES
  • 1. Leaphart et al., 2008, Interferon-gamma inhibits enterocyte migration by reversibly displacing connexin43 from lipid rafts. Am J Physiol Gastrointest Liver Physiol 295: G559-569.
  • 2. Leaphart et al., 2007, Interferon-[gamma] inhibits intestinal restitution by preventing gap junction communication between enterocytes. Gastroenterology 132: 2395-2411.
  • 3. Goodenough, 1974, Bulk isolation of mouse hepatocyte gap junctions. Characterization of the principal protein, connexin, J. Cell Biol. 61:557-563.
  • 4. Goodenough, 1975, The structure of cell membranes involved in intervellular communication. Am. J. Clin. Pathol. 63:636-645.
  • 5. Laird, 2005, Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation. Biochim. Biophys. Acta 1711:172-182.
  • 6. Lampe et al., 2000, Phosphorylation of connexin-43 on serine 368 by protein kinase C regulates gap junction communication. J. Cell Biol. 149:1503-1512.
  • 7. Verma et al., 2009, Novel pharmacophores of connexin43 based on the “RXP” series of Cx43-binding peptides, Circ Res. 105(2):176-84.
Various publications are cited herein, the contents of which are hereby incorporated by reference in their entireties.

Claims (15)

What is claimed is:
1. A method of treating necrotizing enterocolitis in a subject in need of such treatment comprising administering to the subject one or more dose of a gap junction enhancing agent, wherein the one or more dose of the gap junction enhancing agent is between 0.05 mg/kg and 10 mg/kg, and wherein the gap junction enhancing agent is selected from the group consisting of
(a) a peptide comprising a sequence selected from the group consisting of SEQ ID NO:1-31; and
(b) a peptide analog selected from the group consisting of rotigaptide and GAP-134 ((2S,4R)-1-(2-aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic acid).
2. The method of claim 1 where the gap junction enhancing agent comprises a peptide having a sequence selected from the group consisting of SEQ ID NO:1-31.
3. The method of claim 1 where the gap junction enhancing agent is rotigaptide.
4. A method of reducing the risk of occurrence of necrotizing enterocolitis in a subject in need of such treatment comprising administering to the subject one or more dose of a gap junction enhancing agent, wherein the one or more dose of the gap junction enhancing agent is between 0.05 mg/kg and 10 mg/kg, and wherein the gap junction enhancing agent is selected from the group consisting of
(a) a peptide comprising a sequence selected from the group consisting of SEQ ID NO:1-31; and
(b) a peptide analog selected from the group consisting of rotigaptide and GAP-134 ((2S,4R)-1-(2-aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic acid).
5. The method of claim 4 where the gap junction enhancing agent comprises a peptide having a sequence selected from the group consisting of SEQ ID NO:1-31.
6. The method of claim 4 where the gap junction enhancing agent is rotigaptide.
7. A method of inducing increased migration of enterocytes in a subject diagnosed with necrotizing enterocolitis, the method comprising administering to the subject one or more dose of a gap junction enhancing agent, wherein the one or more dose of the gap junction enhancing agent is between 0.05 mg/kg and 10 mg/kg, and wherein the gap junction enhancing agent is selected from the group consisting of
(a) a peptide comprising a sequence selected from the group consisting of SEQ ID NO:1-31; and
(b) a peptide analog selected from the group consisting of rotigaptide and GAP-134 ((2S,4R)-1-(2-aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic acid).
8. The method of claim 7 where the gap junction enhancing agent comprises a peptide having a sequence selected from the group consisting of SEQ ID NO:1-31.
9. The method of claim 7 where the gap junction enhancing agent is rotigaptide.
10. The method of claim 1, wherein the one or more dose of the gap junction enhancing agent is between 0.05 mg/kg and 5 mg/kg.
11. The method of claim 10, wherein the one or more dose of the gap junction enhancing agent is between 0.1 mg/kg and 1 mg/kg.
12. The method of claim 4, wherein the one or more dose of the gap junction enhancing agent is between 0.05 mg/kg and 5 mg/kg.
13. The method of claim 12, wherein the one or more dose of the gap junction enhancing agent is between 0.1 mg/kg and 1 mg/kg.
14. The method of claim 7, wherein the one or more dose of the gap junction enhancing agent is between 0.05 mg/kg and 5 mg/kg.
15. The method of claim 14, wherein the one or more dose of the gap junction enhancing agent is between 0.1 mg/kg and 1 mg/kg.
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